ScFv-Fc dimers that bind transforming growth factor-BETA1 with high affinity, avidity and specificity

• US 10,508,146 B2
• Filed: 03/03/2016
• Issued: 12/17/2019
• Est. Priority Date: 03/04/2015
• Status: Active Grant

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Qiu, Huawei, Pan, Clark, Bird, Julie
• Primary Examiner(s): Xie, Xiaozhen
• Application Number:
  US15/555,499
• Publication Number:
  US 20180222970A1
• Time in Patent Office: 1,384 Days
• Field of Search: None
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/35 Valency
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/626 : Diabody or triabody
• CPC Class Codes: C07K 2317/64 : comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/70 : characterized by effect upon binding to a cell or to an antigen
• CPC Class Codes: C07K 2317/72 : Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/23 : containing a GST-tag
• CPC Class Codes: C07K 2319/73 : containing coiled-coiled motif (leucine zippers)
• CPC Class Codes: C12N 15/11 : DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins

Abstract

An scFv-Fc dimer binds and neutralizes TGFβ1 selectively and with high affinity and avidity. The scFv region may comprise the same VH and VL domains or CDR regions as metelimumab. The unique combination of their smaller size, high selectivity, potency against TGFβ1, and long in vivo half-life makes the scFv-Fc dimers ideal candidates for therapeutic applications.

Claims

1. An isolated binding protein that binds TGFβ

1, wherein the isolated binding protein is a dimer formed from a polypeptide chain having the formula of, from N-terminus to C-terminus;

(VH domain)-(linker1)-(VL domain)-(linker2)-(hinge)-(Fc region),

wherein

the VH domain comprises a heavy chain complementarity determining region (HCDR) 1 having the amino acid sequence of SEQ ID NO;

22, an HCDR2 having the amino acid sequence of SEQ ID NO;

23, and an HCDR3 having the amino acid sequence of SEQ ID NO;

24, 25, 26, or 30; and

the VL domain comprises a light chain complementarity determining region (LCDR) 1 having the amino acid sequence of SEQ ID NO;

27 or the amino acid sequence of SEQ ID NO;

27 with an A2S substitution, an LCDR2 having the amino acid sequence of SEQ ID NO;

28, and an LCDR3 having the amino acid sequences of SEQ ID NO;

29;

the linker1 is a [G₄S]₃-type linker;

the linker2 is SEQ ID NO;

20 or a variant thereof, wherein the variant differs from SEQ ID NO;

20 in length by one to four amino acids, or differs from SEQ ID NO;

20 by having up to two amino acid substitutions from glycine to serine or from serine to glycine; and

the hinge comprises an amino acid sequence from a human IgG₁ or IgG₄ hinge region, or the amino acid sequence of SEQ ID NO;

7 or 21.

2. The isolated binding protein of claim 1, wherein

the VH domain comprises the human VH domain sequence set forth in SEQ ID NO:

1 or 2, or a variant thereof having up to four amino acid modifications; and

the VL domain comprises the human Vκ

domain sequence set forth in SEQ ID NO;

5 or 6, or a variant thereof having up to four amino acid modifications.

3. The isolated binding protein of claim 2, wherein the VH and VL domains comprise the amino acid sequences set forth in SEQ ID NOs:

1 and 5, respectively.

4. The isolated binding protein of claim 1, wherein the linker1

comprises the amino acid sequence SGGGSGGGGSGGGGS (SEQ ID NO:

3) or GGGGSGGGGSGGGGS (SEQ ID NO;

     4).

5. The isolated binding protein of claim 1, wherein the linker2 is SEQ ID NO:

20.

6. The isolated binding protein of claim 1, wherein the Fc region is derived from a human IgG₁, a human IgG₄, or a variant of a human IgG₁ or IgG₄ wherein up to ten amino acids are modified.

7. The isolated binding protein of claim 1, wherein the polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

9.

8. The isolated binding protein of claim 1, wherein the isolated binding protein has at least one of the following characteristics:

a) binding selectively to TGFβ

1;

b) having an IC₅₀ to human TGFβ

1 of less than 1 nM in an A549 bioassay;

c) exhibiting a Kd for human TGFβ

1 at least about 50% lower than a Kd for human TGFβ

2 as measured by surface plasmon resonance; and

d) exhibiting a Kd for human TGFβ

1 at least about 50% lower than a Kd for human TGFβ

3 as measured by surface plasmon resonance.

9. A pharmaceutical composition comprising the isolated binding protein of claim 1 and a pharmaceutically acceptable excipient.

10. A method of treating a disease in a human in need of inhibition of TGFβ

1 activity, comprising administering to the human a therapeutically effective amount of the isolated binding protein of claim 1.

11. The method of claim 10, wherein the disease is selected from the group consisting of a fibrotic disease, cancer, and an immune-mediated disease.

12. The method of claim 10, wherein the disease is diffuse cutaneous systemic sclerosis, bone remodeling disease, or kidney disease.